PROJECT SUMMARY/ABSTRACT Pancreatic cancers, about 95% of which are pancreatic ductal adenocarcinoma (PDAC), have the worst prognosis of all cancers and will soon be the second leading cause of cancer-related deaths in the United States. Current treatment regimens clearly fail to benefit patient survival. Oncolytic virotherapy is a relatively new anticancer approach that utilizes replication-competent viruses to specifically infect and kill tumor cells. Vesicular stomatitis virus (VSV) is one such oncolytic virus (OV) and is already in several phase I clinical trials against different malignancies. Our previous studies demonstrated that VSV is effective against the majority of tested human PDAC cell lines, both in vitro and in vivo. However, we found that some PDAC cell lines are resistant to VSV infection, replication, and/or virus-mediated oncolysis. Our goal is to better understand and target factors determining the success of VSV-based OV therapy and to develop rational therapy approaches to enhance OV efficacy and overcome resistance of PDACs to OVs. The proposal is based on our recently published work and new preliminary studies that analyzed VSV against a number of PDAC cell lines in vitro and in vivo, made an initial characterization of PDAC cells lines in regard to their general cellular characteristics and permissiveness to VSV, and identified two chemovirotherapy combination approaches dramatically improving VSV performance in PDAC cells. The proposed experiments are based on our preliminary studies screening various FDA-approved chemotherapeutic drugs for their compatibility with VSV for PDAC treatment, and demonstrating that, while most tested drugs inhibited VSV replication in PDAC cells and did not improve oncotoxicity (and thus are not compatible with VSV), two drugs, paclitaxel and gemcitabine, dramatically improved oncotoxicity compared to single treatments. In Aim 1, we will determine in vitro molecular mechanisms responsible for the increased oncotoxicity of VSV/paclitaxel and VSV/gemcitabine combinations. Our preliminary data indicate that the mechanisms of the observed improvements in oncotoxicity are different for these drugs, as paclitaxel strongly increased VSV replication, while gemcitabine inhibited it. in Aim 2, we will examine in vivo the efficacy and safety of these combination treatments using clinically-relevant mouse models of PDAC. Our studies will develop more effective VSV oncolytic therapies and broaden the spectrum of PDAC phenotypes to which this treatment can be successfully applied. In addition, they may lead to development of prescreening approaches for selecting patients responsive to a particular OV-based combination treatment. While this proposal is focused on VSV against PDAC, our discoveries will be relevant to other OVs and tumor systems.